Molecular Recognition Force Spectroscopy for Probing Cell Targeted Nanoparticles In Vitro

In the development and design of cell targeted nanoparticle-based systems the density of targeting moieties plays a fundamental role in allowing maximal cell-specific interaction. Here, we describe the use of molecular recognition force spectroscopy as a valuable tool for the characterization and optimization of targeted nanoparticles toward attaining cell-specific interaction. By tailoring the density of targeting moieties at the nanoparticle surface, one can correlate the unbinding event probability between nanoparticles tethered to an atomic force microscopy tip and cells to the nanoparticle vectoring capacity. This novel approach allows for a rapid and cost-effective design of targeted nanomedicines reducing the need for long and tedious in vitro tests.The authors would like to acknowledge the Bioimaging Platform (i3S-INEB) for the support with atomic force microscopy. This work was financed by projects NORTE-01-0145-FEDER-000008 and NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the projects UID/BIM/04293/ 2013, PTDC/CTM-NAN/115124/2009, and PTDC/CTMNAN/3547/2014. C.P. Gomes acknowledge FCT for her PhD scholarship SFRH/BD/79930/2011

Novel “nano-phage” interfaces for wireless biosensors

The prevention of food-borne illness has become a very important factor in public health. Meantime, the conventional microbiological detection techniques are time consuming, require proficiency and appropriate laboratory conditions. Recently, there has been an extensive work undertaken towards the development of diagnostic biosensor devices for on-site detection of biological threats that explore a diversity of transduction mechanisms and bio-recognition elements. In particular, the environmentally robust filamentous phages have been successfully used as an alternative to fragile antibodies in wireless biosensor system for real-time pathogen detection. However, when phages are used as interface, they can aggregate forming bundles of fibers that cannot cover completely the sensor’s interface leading to the decrease in sensor’s performance. In this work we developed novel wireless magnetoelastic biosensors with interface formed by biorecognition nanoparticles called “nano-phage”. “Nano-phage” comprises nanoparticles with diameter ~11 nm composed of self-assembled fusion major coat protein of landscape phages selected against the target analyte. For proof-of-concept, we investigated interfaces formed by three model phages selected from landscape libraries: streptavidin binders 7b1 and SAE10 and clone E2 highly specific and selective for S. typhimurium. Beside food borne pathogens, this new approach can be used to develop biosensors with increased performance for early detection of cancer diseases and other pathologies

Atomic Force Microscopy as a Tool to Assess the Specificity of Targeted Nanoparticles in Biological Models of High Complexity

The ability to design nanoparticle delivery systems capable of selectively target their payloads to specific cell populations is still a major caveat in nanomedicine. One of the main hurdles is the fact that each nanoparticle formulation needs to be precisely tuned to match the specificities of the target cell and route of administration. In this work, molecular recognition force spectroscopy (MRFS) is presented as a tool to evaluate the specificity of neuron-targeted trimethyl chitosan nanoparticles to neuronal cell populations in biological samples of different complexity. The use of atomic force microscopy tips functionalized with targeted or non-targeted nanoparticles made it possible to assess the specific interaction of each formulation with determined cell surface receptors in a precise fashion. More importantly, the combination of MRFS with fluorescent microscopy allowed to probe the nanoparticles vectoring capacity in models of high complexity, such as primary mixed cultures, as well as specific subcellular regions in histological tissues. Overall, this work contributes for the establishment of MRFS as a powerful alternative technique to animal testing in vector design and opens new avenues for the development of advanced targeted nanomedicines.The authors would like to acknowledge the Bioimaging Center for Biomaterials and Regenerative Therapies of INEB (b.IMAGE) for the support with atomic force and confocal microscopy, Centro de Materiais da Universidade do Porto (CEMUP) for NMR analysis and the Biointerfaces and Nanotechnology Service (INEB-i3S) for nanoparticle size and zeta-potential analysis. The work was financed by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the projects UID/BIM/04293/2013, PTDC/CTM-NAN/115124/2009, and PTDC/CTM-NAN/3547/2014. C.P.G. and C.D.F.L. acknowledge FCT for their Ph.D. scholarships SFRH/BD/79930/2011 and SFRH/BD/77933/2011, respectively

Force-Sensitive Autoinhibition of the von Willebrand Factor ls Mediated by Interdomain Interactions

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis

Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein

<p>Abstract</p> <p>Background</p> <p>Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure.</p> <p>Results</p> <p>Avidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (K_d~ 9 μM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between <it>β</it>-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins.</p> <p>Conclusions</p> <p>The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.</p

pH-dependent deformations of the energy landscape of avidin-like proteins investigated by single molecule force spectroscopy

Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very high pH values. We compared avidin with streptavidin and a recently developed avidin-based mutant, chimeric avidin. To gain insights of the energy landscape of these interactions we used a single molecule approach and performed the Single Molecule Force Spectroscopy atomic force microscopy technique. There, the ligand (biotin) is covalently coupled to a sharp AFM tip via a distensible hetero-bi-functional crosslinker, whereas the receptor of interest is immobilized on the probe surface. Receptor-ligand complexes are formed and ruptured by repeatedly approaching and withdrawing the tip from the surface. Varying both pulling velocity and pH value, we could determine changes of the energy landscape of the complexes. Our results clearly demonstrate that avidin, streptavidin and chimeric avidin are stable over a wide pH range although we could identify differences at the outer pH range. Taking this into account, they can be used in a broad range of applications, like surface sensors at extreme pH values

Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA1 D130E, HA2 I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production

Probing Specific Interaction Forces Between Human IgG and Rat Anti-Human IgG by Self-Assembled Monolayer and Atomic Force Microscopy

Interaction forces between biological molecules such as antigen and antibody play important roles in many biological processes, but probing these forces remains technically challenging. Here, we investigated the specific interaction and unbinding forces between human IgG and rat anti-human IgG using self assembled monolayer (SAM) method for sample preparation and atomic force microscopy (AFM) for interaction force measurement. The specific interaction force between human IgG and rat anti-human IgG was found to be 0.6–1.0 nN, and the force required for unbinding a single pair of human IgG and rat anti-human IgG was calculated to be 144 ± 11 pN. The results are consistent with those reported in the literatures. Therefore, SAM for sample preparation combined with AFM for interaction measurement is a relatively simple, sensitive and reliable technique to probe specific interactions between biological molecules such as antigen and antibody

Dynamic force microscopy for imaging of viruses under physiological conditions

Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized